Project Abstract This SHINE II application seeks to explore epigenetic mechanisms that control globin gene regulation during erythropoiesis. The central hypothesis of this project is that that miR-29b reverses the ?-globin to ?-globin gene switch and induces HbF via modulation of DNA methylation. Therapeutic interventions aimed at inducing HbF expression is an effective approach for ameliorating the clinical symptoms of sickle cell disease (SCD) in adults and children. Hydroxyurea is the only FDA-approved drug with proven efficacy for inducing HbF in patients with SCD, but DNA methyltransferase (DNMT) inhibitors have shown promise as HbF inducers by producing proximal ?-globin promoter DNA hypomethylation. However, DNMT inhibitors can produce off-target side effects. Small non-coding microRNAs (miR) are attractive molecules for targeting repressors of ?-globin gene expression and studies show that miR-29b inhibits DNA methylation through direct targeting of the 3? untranslated region of DNMT3A and DNMT3B. The objective of this proposal is to test the efficacy of miR-29b as an HbF inducer. Our published work shows that miR-29b is important for reactivating ?-globin transcription and HbF expression by targeting MYB, a known repressor of ?-globin involved in mediating DNA methylation and gene silencing. To test our central hypothesis, we will accomplish one specific aim to determine the ability of miR-29b to mediate epigenetic changes in DNA methylation in the HBB locus and reactivate ?-globin transcription and HbF expression during adult erythropoiesis. Sub-aim A will explore the molecular effects of miR-29b on ?-globin regulation using an in vitro primary liquid culture system to generate erythroid progenitors from peripheral blood mononuclear cells isolated from individuals with SCD. These findings will compare to normal erythroid progenitors to validate the ability of miR-29b to reactivate ?-globin transcription through proximal promoter DNA methylation. Off-target effects mediated by miR-29b will be investigated by DNA mutation and RNA-seq analyses. To define the role of miR-29b in reversing phenotype, erythroid progenitor sickling under hypoxic conditions will be evaluated. Sub-aim B will establish the optimal dose of miR-29b that mediates ?-globin reactivation and HbF induction in vivo using a preclinical Townes SCD mouse model. This research highlights a novel miRNA-based epigenetic approach to induce HbF to impact the discovery of new drugs to expand treatment options for SCD.